Tumor suppressor gene, p47Ing3

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of novel human tumor suppressors, antibodies to such tumor suppressors, methods of detecting such nucleic acids and proteins, methods of screening for modulators of tumor suppressors, and methods of diagnosing and treating tumors with such nucleic acids and proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patentapplication Serial No. 60/181,292, filed on Feb. 9, 2000, the teachingsof which are herein incorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] This invention relates to isolated nucleic acid and amino acidsequences of novel human tumor suppressors, antibodies to such tumorsuppressors, methods of detecting such nucleic acids and proteins,methods of screening for modulators of tumor suppressors, and methods ofdiagnosing and treating tumors with such nucleic acids and proteins.

BACKGROUND OF THE INVENTION

[0004] Certain tumors, benign, premalignant, and malignant, are known tohave genetic components. Mutations or inactivation of “tumor suppressor”genes causes some of these tumors. In normal cells, the tumor suppressorgenes are involved in the regulation of cell growth and proliferationand in the control of cellular aging, anchorage dependence andapoptosis. When the tumor suppressor genes are mutated or inactivated,cells are transformed and become immortalized or tumorigenic. Thesetransformed cells can be reverted back to the normal phenotype (i.e.,the cell growth rate is suppressed) by introducing the wildtypesuppressor genes.

[0005] The first tumor suppressor gene identified was the nuclearphosphoprotein, retinoblastoma gene (Rb). Retinoblastoma is a malignanttumor of the sensory layer of the retina, and often occurs bilaterallyduring childhood. Retinoblastoma exhibits a familial tendency, but itcan be acquired. Mutations in the Rb gene and inactivation of itsproduct have been shown to be involved in other tumors, such as bladder,breast, and small cell lung carcinomas, osteosarcomas, and soft tissuesarcomas. It was demonstrated that reconstitution of Rb-deficient tumorcells with the wildtype Rb leads to the suppression of growth rate ortumorigenicity (Huang et al., Science 242:1563-1566 (1988)). This resultprovides direct evidence that Rb protein is a tumor suppressor.

[0006] Another well-characterized tumor suppressor is the gene for thenuclear phosphoprotein, p53. More than half of all human cancers areassociated with mutations in the tumor suppressor gene p53 (see, e.g.,Hollstein et al., Science 253:49-53 (1991); Caron de Fronmentel &Soussi, Genes Chromosom. Cancer 4: 1-15; Harris & Hollstein, N. Engl. J.Med. 329:1318-1327 (1993); Greenblatt et al., Cancer Res. 54:4855-4878(1994)). Mutations in p53 often appear to be a critical step in thepathogenesis and progression of tumors. For example, missense mutationsof p53 occur in tumors of the colon, lung, breast, ovary, bladder, andseveral other organs. Alternatively, inactivation of the wildtype p53proteins in cells can cause tumors. For example, certain strains ofhuman papillomavirus (HPV) are known to interfere with the p53 proteinfunction, because the virus produces a protein, E6, which promotes thedegradation of the p53 protein.

[0007] Recently, another tumor suppressor gene, p33ING1, has beenidentified. p33ING1 directly cooperates with tumor suppressor gene p53in growth regulation (Garkavtsev et al., Nature Genetics 14:415420(1996); Garkavtsev et al., Nature 391:295-298 (1998); GenBank AccessionNo. AF044076; SEQ ID NO: 8). Neither of the two genes can alone causegrowth inhibition when the other one is suppressed (Garkavtsev et al.(1998), supra). According to immunoprecipitation studies, p33ING1proteins modulate the p53 activity through physical interaction. It hasbeen also reported that some neuroblastoma cells have a mutation of thep33ING1 gene, and some breast cancer cell lines exhibit reducedexpression of p33ING1 (Garkavtsev et al. (1996), supra). A tumorsuppressor gene with homology to p33ING1, p33ING2, has also been clonedand characterized (See Harris and Nagashima, U.S. Provisional PatentApplication No. 60/121,891, filed on Feb. 26, 1999; SEQ ID NOS: 6 and7).

[0008] Cancer remains a major public concern. Although epidemiologicaland cytogenetic studies demonstrated that a number of recessive geneticmutations are involved in various cancers, only a limited number oftumor suppressors have been identified. Therefore, there is a need toidentify and isolate other tumor suppressor genes. The identificationand isolation of new tumor suppressor genes would allow aid indiagnosis, prevention, and treatment of tumors and cancers.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for identifying acompound that modulates p47ING3, the method comprising the steps of: (i)contacting the compound with a eukaryotic host cell or cell membrane inwhich has been expressed a tumor suppressor polypeptide (p47ING3), thepolypeptide: (a) having greater than about 70% amino acid sequenceidentity to a polypeptide having a sequence of SEQ ID NO:1; and, (b)selectively binding to polyclonal antibodies generated against SEQ IDNO:1; and (ii) determining the functional effect of the compound uponthe cell or cell membrane expressing the polypeptide.

[0010] In one embodiment, the functional effect is determined bymeasuring changes in cell growth.

[0011] In another aspect, the present invention provides a method ofinhibiting cellular proliferation, the method comprising transducing acell with an expression vector, the vector comprising a nucleic acidencoding a tumor suppressor polypeptide (p47ING3), the polypeptide: (i)having greater than about 70% amino acid sequence identity to apolypeptide having a sequence of SEQ ID NO:1; and, (ii) selectivelybinding to polyclonal antibodies generated against SEQ ID NO:1.

[0012] In another aspect, the present invention provides a method ofdetecting the presence or absence of p47ING3 in tumorigenic mammaliantissue, the method comprising the steps of: (i) isolating a tumorigenicsample; (ii) contacting the tumorigenic sample with a p47ING3-specificreagent that selectively associates with p47ING3; and (iii) detectingthe level of p47ING3-specific reagent that selectively associates withthe tumorigenic sample.

[0013] In one embodiment, the p47ING3-specific reagent is selected fromthe group consisting of a p47ING3-specific antibody, a p47ING3-specificprimer; and a p47ING3-specific nucleic acid probe.

[0014] In another aspect, the present invention provides a for anisolated nucleic acid encoding a tumor suppressor polypeptide (p47ING3),the nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 2.

[0015] Also, the present invention provides an expression vectorcomprising the nucleic acid of SEQ ID NO: 2. In one embodiment, a hostcell is transfected with an expression vector comprising the nucleicacid of SEQ ID NO:2.

[0016] In another aspect, the present invention provides an isolatedtumor suppressor polypeptide (p47ING3), the polypeptide comprising anamino acid sequence of SEQ ID NO: 1.

[0017] The present invention further provides an antibody thatselectively binds to a p47ING3 polypeptide of SEQ ID NO: 1. In oneembodiment, the antibody is polyclonal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 illustrates binding specificities of polyclonal antibodiesfor p47ING3 raised against SEQ ID NO: 5 by Western blot analysis. Themolecular sizes of standards are indicated in kDa on the left handborder of the gel. The proteins were produced using the TNT T7 QuickCoupled Transcription/Translation System, Cat. # L1170 from PromegaCorporation of Madison, Wis.

[0019]FIG. 2 illustrates binding specificities of polyclonal antibodiesfor p47ING3 raised against SEQ ID NO: 9 by Western blot analysis. Themolecular sizes of standards are indicated in kDa on the right handborder of FIG. 2. The proteins were produced using the TNT T7 QuickCoupled Transcription/Translation System, Cat. # L1170 from PromegaCorporation of Madison, Wis. The proteins were electrophoresed andWestern blotted. The Western blot was incubated with an anti-p47ING3antibody (diluted 1:200) raised against SEQ ID NO: 9. The presence ofthe p47ING3 immunoreactive bands was visualized with a goat anti-rabbitIgG-HRP (1:2000 dilution) (Santa Cruz Biotechnology) and ECL WesternBlotting Detection Reagents (Amersham Pharmacia Biotech, RPN2106). Theblot was then subjected to autoradiography using Hyperfilm ECL (AmershamPharmacia Biotech, RPN2103K).

[0020]FIG. 3 illustrates that p47ING3 inhibits the cell growth of thehuman colon carcinoma RKO cell line by colony formation assay. The lefthand panel shows RKO cells transfected with the parent vector pcDNA3.1and the right hand panel shows RKO cells transfected with a vector thatencodes for p47ING3, pcDNA3.1-p47ING3.

[0021]FIG. 4 shows a FACS analysis of the RKO (human colon carcinoma)cells transfected with pcDNA3.1 (top panel) or pcDNA3.1-p47ING3 (bottompanel). The cells were co-transfected with the plasmid pEGFP-F. Thecells were stained with propidium iodide. The cells were then gated forGFP fluorescence using FACscan.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] I. Introduction

[0024] The present invention provides for the first time nucleic acidsand polypeptides of a new tumor suppressor called p47ING3. The presentinvention also provides antibodies which specifically hybridize to ap47ING3 protein. These nucleic acids and the polypeptides they encodeare tumor suppressors that are involved in the regulation of cellproliferation and in the control of cellular aging, anchoragedependence, and apoptosis.

[0025] The present invention also provides methods of screening formodulators (e.g., activators, inhibitors, stimulators, enhancers,agonists, and antagonists) of these novel p47ING3 proteins. Suchmodulators are useful for pharmacological and genetic modulation of cellgrowth and tumor suppression. The invention thus provides assays fortumor suppression and cell growth, where p47ING3 acts as a direct orindirect reporter molecule for measuring the effect of modulators oncell growth or tumor suppression. These assays can measure variousparameters that are affected by the p47ING3 activity, e.g., cell growthon soft agar, contact inhibition and density limitation of growth,growth factor or serum dependence, tumor specific markers levels,invasiveness into Matrigel, tumor growth in vivo, arrest of cells inG₀/G₁ phase of the cell-cycle, p47ING3 protein or mRNA levels,transcriptional activation or repression of a reporter gene, and thelike.

[0026] The present invention also provides methods of inhibiting cellproliferation of a cell by transducing the cell with an expressionvector containing p47ING3 nucleic acids. The transduced cell may have amissense or null endogenous p47ING3 phenotype or a mutation in anothertumor suppressor gene. Expression of wildtype p47ING3 restores cellgrowth regulation and prevents the development of tumor. For example,p47ING3 nucleic acids can be used to treat cancer or other cellproliferative diseases, such as hyperplasia, in patients.

[0027] Finally, the invention provides for methods of detecting p47ING3or nucleic acid and protein expression, allowing investigation of cellgrowth regulation and tumor suppression. Furthermore, p47ING3 nucleicacid and protein expression can be used to diagnose cancer in patientswho have a defect in one or more copies of p47ING3 in their genome.

[0028] Functionally, p47ING3 represents a protein having a molecularweight of approximately 40-47 kDa. It is involved in the regulation ofcell proliferation and in the control of cellular aging, anchorage andapoptosis.

[0029] Structurally, the nucleotide sequence of p47ING3 (see, e.g. SEQID NO:2, isolated from a human) encodes a polypeptide of approximately418 amino acids with a predicted molecular weight of approximately 47kDa (see, e.g., SEQ ID NO:1). Related p47ING3 genes from other speciesshare at least about 70% amino acid identity over an amino acid regionof at least about 25 amino acids in length, preferably 50 to 100 aminoacids in length.

[0030] Specific regions of the p47ING3 nucleotide and amino acidsequences may be used to identify polymorphic variants, interspecieshomologs, and alleles of p47ING3. This identification can be made invitro, e.g., under stringent hybridization conditions or with PCR andsequencing, or by using the sequence information in a computer systemfor comparison with other nucleotide or amino acid sequences. Typically,identification of polymorphic variants and alleles of p47ING3 is made bycomparing an amino acid sequence of about 25 amino acids or more,preferably 50-100 amino acids. Amino acid identity of approximately atleast 70% or above, preferably 80%, most preferably 90-95% or abovetypically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of p47ING3. Sequence comparison can beperformed using any of the sequence comparison algorithms discussedbelow. Antibodies that bind specifically to p47ING3 or a conservedregion thereof can also be used to identify alleles, interspecieshomologs, and polymorphic variants.

[0031] Polymorphic variants, interspecies homologs, and alleles ofp47ING3 are conformed by examining the effect of putative p47ING3expression on cell growth and tumor suppression using the methods andassays described herein. Typically, p47ING3 having the amino acidsequence of SEQ ID NO: 1 is used as a positive control. For example,immunoassays using antibodies directed against the amino acid sequenceof SEQ ID NOS: 1, 5, or 9 can be used to demonstrate the identificationof a polymorphic variant or allele of p47ING3. Alternatively, p47ING3having the nucleic acid sequences of SEQ ID NO:2 is used as a positivecontrol, e.g., in in situ hybridization with SEQ ID NO:2 to demonstratethe identification of a polymorphic variant or allele of p47ING3. Thepolymorphic variants, alleles and interspecies homologs of p47ING3 areexpected to retain the ability to inhibit cell proliferation and tumorsuppression. These functional characteristics can be tested usingvarious assays, such as soft agar assay, contact inhibition and densitylimitation of growth assay, growth factor or serum dependence assay,tumor specific markers assay, invasiveness assay, tumor growth assay,etc.

[0032] p47ING3 nucleotide and amino acid sequence information may alsobe used to construct models of tumor suppressor polypeptides in acomputer system. These models are subsequently used to identifycompounds that can activate or inhibit p47ING3. Such compounds thatmodulate the activity of p47ING3 can be used to investigate the role ofp47ING3 in inhibition of cell proliferation and tumor suppression or canbe used as therapeutics.

[0033] Isolation of p47ING3 provides a means for assaying for modulatorsof p47ING3. p47ING3 is useful for testing modulators using in vivo andin vitro expression that measure various parameters, e.g., cell growthon soft agar, contact inhibition and density limitation of growth,growth factor or serum dependence, tumor specific markers levels,invasiveness into Matrigel, tumor growth in vivo, p47ING3 protein ormRNA levels, transcriptional activation or repression of a reportergene, and the like. Such modulators identified using p47ING3 can be usedto study cell growth regulation and tumor suppression, and further totreat cancer.

[0034] Methods of detecting p47ING3 nucleic acids and expression ofp47ING3 are also useful for to diagnose various cancers or tumors byusing assays such as northern blotting, dot blotting, in situhybridization, RNase protection, and the like. Chromosome localizationof the genes encoding human p47ING3 can also be used to identifydiseases, mutations, and traits caused by and associated with p47ING3.Techniques, such as high density oligonucleotide arrays (GeneChip™,Affymetrix), can be also be used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of p47ING3.

[0035] II. Definitions

[0036] As used herein, the following terms have the meanings ascribed tothem unless specified otherwise.

[0037] The term “tumor suppressor” refers to a gene, or the protein itencodes, that in its wildtype form has the ability to suppress, prevent,or decrease cell transformation. Tumor suppressor ones are genes thatencode protein(s) that regulate cell growth and proliferation directlyor indirectly, e.g., p53, Rb, and the like. If a tumor suppressor geneis damaged (e.g., by radiation, a carcinogen or inherited, orspontaneous mutation), it may lose its wildtype ability to regulate cellgrowth and proliferation, and the cells may become transformed orpre-disposed to transformation.

[0038] “p47ING” refers to a family of tumor suppressor nucleic acids orpolypeptides having a molecular weight of approximately 40-47 kDa. Theyencode a nuclear protein which is involved in the regulation of cellgrowth and proliferation and in the control of cellular aging, anchorageand apoptosis.

[0039] The term “p47ING3” therefore refers to polymorphic variants,alleles, interspecies homologs, and mutants that: (1) have about 70%amino acid sequence identity, preferably about 80-90% amino acidsequence identity to SEQ ID NO:1 over a window of about at least 50-100amino acids; (2) binds to polyclonal antibodies raised against animmunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1 and conservatively modified variants thereof,but does not bind to polyclonal antibodies raised against an immunogencomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:8 or SEQ ID NO: 6 and conservatively modified variantsthereof; (3) specifically hybridize (with a size of at least about 500,preferably at least about 900 nucleotides) under stringent hybridizationconditions to a sequence selected from the group consisting of SEQ IDNO:2, and conservatively modified variants thereof; or (4) are amplifiedby primers that specifically hybridize under stringent conditions to thesame sequence as a degenerate primers sets encoding SEQ ID NOS:3 and 4.

[0040] A “test compound is able to modulate a p47ING3 activity” if thetest compound can increase or decrease a property associated withp47ING3.

[0041] A “cell expresses p47ING3 above basal levels” when the cellproduces p47ING3 protein or mRNA in amounts greater than amountsproduced in the parent cell or the untransfected cell. The amount ofp47ING3 protein or mRNA can be determined using methods known in theart, such as Western blots or Northern blots, respectively.

[0042] The term “modulate” refers to an increase or decrease in aparameter that is being measured.

[0043] A “p47ING3 activity” can include, but is not limited to p47ING3mediated cell-cycle arrest, p47ING3 induced change in cell growth,p47ING3 mediated decrease of colony formation. These properties can beassayed by comparing the effect of the test compound on a cell that doesnot express p47ING3 above basal levels with a cell that does expressp47ING3 above basal levels. Examples of assays include, but are notlimited to soft agar assay, cell cycle arrest assay, colony formationassay, contact inhibition and density limitation of growth assay, growthfactor or serum dependence assay, anchorage dependence assay, tumorspecific markers assay, invasiveness assay, tumor growth assay, p47ING3protein and mRNA level assays, transcriptional activation or repressionof a reporter gene assay, and the like, in vitro, in vivo, and ex vivo.

[0044] A “test compound” can essentially be any compound, such as achemotherapeutic, a peptide, a hormone, a nucleic acid and the like.

[0045] The phrases “polymorphic variant” and “allele” refer to forms ofp47ING3 that occur in a population (or among populations) and thatmaintain wildtype p47ING3 activity as measured using one of the assaysdescribed herein.

[0046] The term “mutant” of p47ING3 refers to those mutants which areexperimentally made or those which are found in tumor or cancer cells.Mutants of p47ING3 can be due to, e.g., truncation, elongation,substitution of amino acids, deletion, insertion, or lack of expression(e.g., due to promoter or splice site mutations, etc.). A mutant hasactivity that differs from the activity of wildtype p47ING3 by at leastabout 20% as measured using an assay described herein. For example, amutant of p47ING3 can have a null mutation which results in absence ofnormal gene product at the molecular level or an absence of function atthe phenotypic level. Another example is a missense mutation of p47ING3,where a substitution of amino acid(s) results in a change in theactivity of the protein.

[0047] The phrase “missense or null endogenous p47ING3 phenotype” of acell therefore refers to p47ING3 has a missense or null mutation so thatthe cell has a phenotype (e.g., soft agar growth, contact inhibition anddensity limitation of growth, etc.) which differs from a cell having awildtype p47ING3.

[0048] “p33ING2” and “p33ING1” are members of the “p33ING” family, whichmembers are encoded by different genes (i.e., mapped to differentregions on the chromosome). p33ING2 is mapped to human chromosome 7q31.

[0049] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0050] The term “transfect” or “transduce” refers to any way of gettinga nucleic; acid across a cell membrane, including electroporation,biolistics, injection, plasmid transfection, lipofection, viraltransduction, lipid-nucleic acid complexes, naked DNA, etc

[0051] A “host cell” is a naturally occurring cell or a transformed cellthat contains an expression vector and supports the replication orexpression of the expression vector. Host cells may be cultured cells,explants, cells in vivo, and the like. Host cells may be prokaryoticcells such as E. coli, or eukaryotic cells such as yeast, insect,amphibian, or mammalian cells such as CHO, HeLa, HCT116, RKO cells, andthe like.

[0052] “Tumorigenic sample” as used herein is a sample of biologicaltissue or fluid that contains nucleic acids or polypeptides of p47ING3.The biological tissue comprises cancer cells, transformed cells, atumor, a tumor cell and the like. The fluid comprises a solutionobtained from an animal comprising cancer cells, transformed cells, atumor, tumor cells and the like. Such samples include, but are notlimited to, tissue isolated from humans, mice, and rats. Tumorigenicsamples may also include sections of tissues such as frozen sectionstaken from histological purposes. A tumorigenic sample is typicallyobtained from a eukaryotic organism, such as insects, protozoa, birds,fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog,guinea pig, or rabbit, and most preferably a primate such as chimpanzeesor humans.

[0053] “Tumor cell” refers to precancerous, cancerous, and normal cellsin a tumor.

[0054] “Cancer cells”, “transformed” cells or “tansformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, and/or malignancy (see, Freshney, Culture of Animal Cells aManual of Basic Technique (3^(rd) ed. 1994)).

[0055] “Inhibitors,” “activators,” and “modulators” of p47ING3 refer toinhibitory, activating, or modulatory molecules identified using invitro and in vivo assays for tumor suppression, e.g., ligands, agonists,antagonists, and their homologs and mimetics. Inhibitors are compoundsthat decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate tumor suppression, e.g., antagonists.Activators are compounds that increase, activate, facilitate, enhanceactivation, sensitize or up regulate tumor suppression, e.g., agonists.Modulators are inhibitors and activators and include geneticallymodified versions of p47ING3, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for modulators include,e.g. expressing p47ING3 in cells, applying putative modulator compounds,and then determining the functional effects on inhibition of cellproliferation or tumor suppression. Compounds identified by these assaysare used to modulate tumor suppression effect of p47ING3.

[0056] Samples or assays comprising p47ING3 that are treated with apotential modulator are compared to control samples without theinhibitor, activator, or modulator. Control samples (untreated withinhibitors) are assigned a relative p47ING3 activity value of 100%.Inhibition of p47ING3 is achieved when the p47ING3 activity valuerelative to the control is about 90%, preferably 50%, more preferably25-0%. Activation of p47ING3 is achieved when the p47ING3 activity valuerelative to the control (untreated with activators) is 110%, morepreferably 150%, more preferably 200-500%, more preferably 1000-3000%higher.

[0057] The phrase “changes in cell growth” refers to any change in cellgrowth and proliferation characteristics in vitro or in vivo, such asformation of foci, anchorage independence, semi-solid or soft agargrowth, changes in contact inhibition and density limitation of growth,loss of growth factor or serum requirements, changes in cell morphology,gaining or losing immortalization, gaining or losing tumor specificmarkers, ability to form or suppress tumors when injected into suitableanimal hosts, and/or immortalization of the cell. See, e.g., Freshney,Culture of Animal Cells a Manual of Basic Technique, 3^(rd) ed.(Wiley-Liss, Inc. 1994), pp.231-241, herein incorporated by reference.

[0058] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0059] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

[0060] A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

[0061] The term “operably linked” refers to a functional linkage betweena nucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

[0062] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0063] A “label” is a composition detectable by spectroscopic,photochemical., biochemical., immunochemical., or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available (e.g., the polypeptide of SEQ ID NO:1 can bemade detectable, e.g., by incorporating a radiolabel into the peptide,and used to detect antibodies specifically reactive with the peptide).

[0064] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated p47ING3 nucleic acid is separated from openreading frames that flank the p47ING3 gene and encode proteins otherthan p47ING3. The term “purified” denotes that a nucleic acid

protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

[0065] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0066] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

[0067] The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.

[0068] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group (e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refer tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

[0069] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0070] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences, orwhere the nucleic acid does not encode an amino acid sequence, toessentially identical sequences. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol Chem. 260:2605-2608 (1985);Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Because of thedegeneracy of the genetic code, a large number of functionally identicalnucleic acids encode any given protein. For instance, the codons GCA,GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

[0071] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0072] The following groups each contain amino acids that areconservative substitutions for one another:

[0073] 1) Alanine (A), Glycine (G);

[0074] 2) Serine (S), Threonine (T);

[0075] 3) Aspartic acid (D), Glutamic acid (E);

[0076] 4) Asparagine (N), Glutamine (Q);

[0077] 5) Cysteine (C), Methionine (M);

[0078] 6) Arginine (R), Lysine (K), Histidine (H);

[0079] 7) Isoleucine (I), Leucine (L), Valine (V); and

[0080] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0081] (see, e.g., Creighton, Proteins (1984)).

[0082] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides (i.e., 70% identity)that are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Preferably, the percent identity exists over a region of thesequence that is at least about 25 amino acids in length, morepreferably over a region that is 50 or 100 amino acids in length.

[0083] For sequence comparison, one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

[0084] A “comparison window,” as used herein, includes reference to asegment of contiguous positions selected from the group consisting offrom 20 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad Sci. USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection.

[0085] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)).

[0086] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol.215:403410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length. (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

[0087] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0088] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptides per se or thepolypeptides encoded by the nucleic acids (testers) are immunologicallycross reactive with the antibodies raised against the polypeptide(control) as described below. Thus, a polypeptide is typicallysubstantially identical to a second polypeptide, for example, where thetwo peptides differ only by conservative substitutions. Anotherindication that two nucleic acid sequences are substantially identicalis that the two molecules or their complements hybridize to each otherunder stringent conditions, as described below.

[0089] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0090] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with a wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0091] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0092] A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers or a pool of degenerate primers that encode aconserved amino acid sequence, can then be used as a probe understringent hybridization conditions to isolate the test sequence from acDNA or genomic library, or to identify the test sequence in, e.g., aNorthern or Southern blot. Alternatively, another indication that thesequences are substantially identical is if the same set of PCR primerscan be used to amplify both sequences.

[0093] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0094] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

[0095] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3^(rd) ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

[0096] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0097] An “anti-p47ING3” antibody is an antibody or antibody fragmentthat specifically binds a polypeptide encoded by the p47ING3 gene, cDNA,or a subsequence thereof.

[0098] An “anti-p33ING2” antibody is an antibody or antibody fragmentthat specifically binds a polypeptide encoded by the p33ING2 gene, cDNA,or a subsequence thereof.

[0099] An “anti-p33ING1” antibody is an antibody or antibody fragmentthat specifically binds to a polypeptide encoded by the p33ING1 gene,cDNA, or a subsequence thereof.

[0100] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

[0101] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to p47ING3 atleast two times the background, more typically 10 to 100 timesbackground, and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to a polyclonalantibody under such conditions may require an antibody that is selectedfor us specificity for a particular protein. For example, polyclonalantibodies raised to p47ING3 from a species such as rat, mouse, or humancan be selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with p47ING3 and not with other proteins,such as p33ING1 or p33ING2, except for polymorphic variants and allelesof p47ING3. This selection may be achieved for polyclonal antibodies bysubtracting out antibodies that cross react with p33ING1 or p33ING2. Formonoclonal antibodies, the specificity may be achieved by using ap47ING3 specific antigen to make the hybridomas (e.g., SEQ ID NO: 5 orSEQ ID NO: 9). For identifying p47ING3 variants and alleles from aparticular species such as a human, the selection may be achieved bysubtracting out antibodies that cross-react with p33ING2 or p33ING1molecules, respectively, from other species. For species specificmonoclonal antibodies, a species specific antigen can be used to makethe hybridomas. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

[0102] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0103] “p47ING3-specific reagent” refers to any reagent whichspecifically associates with p47ING3. For example, it can be ap47ING3-specific antibody, a p47ING3-specific primer, or ap47ING3-specific nucleic acid probe.

[0104] III. Isolation of the Gene Encoding p47ING3

[0105] A. General Recombinant DNA Methods

[0106] p47ING3 polypeptides and nucleic acids are used in the assaysdescribed below. For example, recombinant p47ING3 can be used to makecells that constitutively express p47ING3. Such polypeptides and nucleicacids can be made using routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2^(nd) ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

[0107] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0108] Oligonucleotides can be chemically synthesized according to thesolid phase phosphoramidite triester method first described by Beaucage& Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Purification of oligonucleotides is typically byeither native acrylamide gel electrophoresis or by anion-exchange HPLCas described in Pearson & Reanier, J. Chrom. 255:137-149 (1983). Thesequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981). Again, as noted above, companies such as Operon Technologies,Inc. provide an inexpensive commercial source for essentially anyoligonucleotide.

[0109] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding p47ING3

[0110] In general., the nucleic acid sequences encoding genes ofinterest, such as p47ING3 and related nucleic acid sequence homologs,are cloned from cDNA and genomic DNA libraries by hybridization with aprobe, or isolated using amplification techniques with oligonucleotideprimers. Preferably mammalian, more preferably human sequences are used.For example, p47ING3 sequences are typically isolated from mammaliannucleic acid (genomic or cDNA) libraries by hybridizing with a nucleicacid probe, the sequence of which can be derived from SEQ ID NO:2 Asuitable tissue from which human p47ING3 RNA and cDNA can be isolatedis, e.g., placenta, heart, brain, placenta, lung, liver, skeletalmuscle, kidney, pancreas, spleen, thyroid, prostate, testis, ovary,small intestine, colon, peripheral blood cell, leukocyte. Heart, Brain,placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,thyroid, prostate, testis, ovary, small intestine, colon, peripheralblood cell, leukocyte

[0111] Amplification techniques using primers can also be used toamplify and isolate, e.g., a nucleic acid encoding p47ING3, from DNA orRNA (see, e.g. Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual(1995)). These primers can be used, e.g., to amplify either the fulllength sequence or a probe of one to several hundred nucleotides, whichis then used to screen a mammalian library for the full-length nucleicacid of choice. For example, degenerate primer sets for p47ING3sequences such as MLYLEDY (SEQ ID NO:3) and RRGSRHK (SEQ ID NO:4) can beused to isolate p47ING3 nucleic acids. Nucleic acids can also beisolated from expression libraries using antibodies as probes. Suchpolyclonal or monoclonal antibodies can be raised, e.g. using thesequence of p47ING3.

[0112] Polymorphic variants and alleles that are substantially identicalto the gene of choice can be isolated using nucleic acid probes, andoligonucleotides under stringent hybridization conditions, by screeninglibraries. Alternatively, expression libraries can be used to clone,e.g., p47ING3 and p47ING3 polymorphic variants, interspecies homologs,and alleles, by detecting expressed homologs immunologically withantisera or purified antibodies made against p47ING3, which alsorecognize and selectively bind to the p47ING3 homolog.

[0113] To make a cDNA library, one should choose a source that is richin the mRNA of choice, e.g., for human p47ING3 mRNA, human coloncarcinoma cell line RKO. The mRNA is then made into cDNA using reversetranscriptase, ligated into a recombinant vector, and transfected into arecombinant host for propagation, screening and cloning. Methods formaking and screening cDNA libraries are well known (see, e.g., Gubler &Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,supra).

[0114] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in non-lambdaexpression vectors. These vectors are packaged in vitro. Recombinantphage are analyzed by plaque hybridization as described in Benton &Davis, Science 196:180-182 (1977). Colony hybridization is carried outas generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA.,72:3961-3965 (1975).

[0115] An alternative method of isolating a nucleic acid and itshomologs combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of, e.g., p47ING3 directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify p47ING3 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of p47ING3 encoding mRNA in physiologicalsamples, for nucleic acid sequencing, or for other purposes. Genesamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

[0116] As described above, gene expression of p47ING3 can also beanalyzed by techniques known in the art, e.g., reverse transcription andPCR amplification of mRNA, isolation of total RNA or poly A+ RNA,northern blotting, dot blotting, in situ hybridization, RNaseprotection, probing high density oligonucleotides, and the like. All ofthese techniques are standard in the art.

[0117] Synthetic oligonucleotides can be used to construct recombinantgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and non-sense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the p47ING3 nucleic acid. Thespecific subsequence is then ligated into an expression vector.

[0118] The nucleic acid encoding the protein of choice is typicallycloned into intermediate vectors before transformation into prokaryoticor eukaryotic cells for replication and/or expression. Theseintermediate vectors are typically prokaryote vectors, e.g., plasmids,or shuttle vectors. Optionally, cells can be transfected withrecombinant p47ING3 operably linked to a constitutive promoter, toprovide higher levels of p47ING3 expression in cultured cells.

[0119] C. Expression in Prokaryotes and Eukaryotes

[0120] To obtain high level expression of a cloned gene or nucleic acid,such as those cDNAs encoding p47ING3, one typically subclones p47ING3into an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation.

[0121] 1. Prokaryotic Expression

[0122] Suitable bacterial promoters are well known in the art anddescribed, e.g., in Sambrook et al. and Ausubel et al. Bacterialexpression systems for expressing the p47ING3 protein are available in,e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene22:229-235 (1983)). Kits for such expression systems are commerciallyavailable. Eukaryotic expression systems for mammalian cells, yeast, andinsect cells are well known in the art and are also commerciallyavailable.

[0123] 2. Eukaryotic Expression

[0124] A variety of methods are known in the art for expressing a genein eukaryotes (Ausubel et al.). These methods often achieve expressionof a gene above basal.

[0125] a. Transfection of Cells with an Expression Cassette.

[0126] Cells can be transfected with an expression cassette containingthe gene of interest and a promoter. The promoter used to directexpression of a heterologous nucleic acid depends on the particularapplication. The promoter is preferably positioned about the samedistance from the heterologous transcription start site as it is fromthe transcription start site in its natural setting. As is known in theart, however, some variation in this distance can be accommodatedwithout loss of promoter function. The promoter typically cam alsoinclude elements that are responsive to transactivation, e.g., hypoxiaresponsive elements, Gal4 responsive elements, lac repressor responsiveelements, and the like. The promoter can be constitutive or inducible,heterologous or homologous.

[0127] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the nucleic acidin host cells. A typical expression cassette thus contains a promoteroperably linked, e.g., to the nucleic acid sequence encoding p47ING3,and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence may typically be linked to a cleavable signal peptide sequenceto promote secretion of the encoded protein by the transformed cell.Such signal peptides would include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

[0128] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0129] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation, e.g.c-myc.

[0130] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

[0131] Some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. Alternatively, high yield expressionsystems not involving gene amplification are also suitable, such asusing a baculovirus vector in insect cells, with a p47ING3 encodingsequence under the direction of the polyhedrin promoter or other strongbaculovirus promoters.

[0132] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical., any ofthe many resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0133] Standard transfection methods are used to produce bacterial.,mammalian, yeast or insect cell lines that express large quantities ofprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

[0134] Any of the well known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook et al., supra). It is only necessarythat the particular genetic engineering procedure used be capable ofsuccessfully introducing at least one gene into the host cell capable ofexpressing the protein of choice.

[0135] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe p47ING3 protein, which is recovered from the culture using standardtechniques identified below.

[0136] b. Gene Activation

[0137] The method of gene activation can also be used to express anendogenous gene, e.g. p47ING3, above basal levels in a cell. Details ofthis technology can be found in U.S. Pat. No. 5,272,071, U.S. Pat. No.5,641,670, EP 0747485B1, EP 0505500B1). Instead of transfecting anexogenous gene in an expression cassette into a cell, these methods relyon the introduction of nucleotide sequences into a cell that willactivate the endogenous gene. The nucleotide sequences are homologouslyrecombined into the cell's genome and cause an increase in thetranscription of the endogenous gene.

[0138] One method involves the activation of a gene that is usuallytranscriptionally silent in the genome of a eukaryotic cell line (U.S.Pat. No. 5,641,670). Briefly, the method involves introducing apolynucleotide sequence containing a targeting sequence, a regulatorysequence, an exon, and an unpaired splice donor site. The polynucleotidesequence is introduced into the cell and homologously recombined withthe endogenous gene. The homologous recombination event permits thepolynucleotide sequence to be operably linked with the endogenous gene.The regulatory sequence is able to promote the transcription of thenormally silent endogenous gene and expression of the gene is achievedabove basal levels.

[0139] The regulatory sequence can contain one or more promoters. Avariety of promoters can be used, such as sequences that regulated theexpression of viral genes, actin genes, immunoglobulin genes and thelike. The regulatory sequences can contain binding sites fortranscription factors, which serve to promote transcription of the genethat is operably linked to the polynucleotide sequence.

[0140] IV. Purification of p47ING3

[0141] If necessary, naturally occurring or recombinant proteins can bepurified for use in functional assays, e.g., to make antibodies todetect p47ING3. Naturally occurring p47ING3 can be purified, e.g., frommammalian tissue such as heart, brain, placenta, lung, liver, skeletalmuscle, kidney, pancreas, spleen, thyroid, prostate, testis, ovary,small intestine, colon, peripheral blood cells, and leukocytes.

[0142] Recombinant p47ING3 is purified from any suitable expressionsystem, e.g., by expressing p47ING3 in E. coli and then purifying therecombinant protein via affinity purification, e.g., by using antibodiesthat recognize a specific epitope on the protein or on part of thefusion protein, or by using glutathione affinity gel, which binds toGST. In some embodiments, the recombinant protein is a fusion protein,e.g., with GST or Gal4 at the N-terminus.

[0143] The protein of choice may be purified to substantial purity bystandard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g. Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

[0144] A number of procedures can be employed when recombinant proteinis being purified. For example, proteins having established molecularadhesion properties can be reversibly fused to p47ING3. With theappropriate ligand, p47ING3 can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,p47ING3 could be purified using immunoaffinity columns.

[0145] A. Purification of p47ING3 from Recombinant Bacteria

[0146] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0147] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g. by incubation ina buffer of 50 mM Tris/HCl pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

[0148] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.The protein of choice is separated from other bacterial proteins bystandard separation techniques, e.g., with Ni-NTA agarose resin.

[0149] Alternatively, it is possible to purify the recombinant p47ING3protein from bacteria periplasm. After lysis of the bacteria, when theprotein is exported into the periplasm of the bacteria, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0150] B. Standard Protein Separation Techniques for Purifying p47ING3

[0151] Solubility Fractionation

[0152] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0153] Size Differential Filtration

[0154] The molecular weight of the protein, e.g., p47ING3, can be usedto isolated it from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

[0155] Column Chromatography

[0156] The protein of choice can also be separated from other proteinson the basis of its size, net surface charge, hydrophobicity, andaffinity for ligands. In addition, antibodies raised against proteinscan be conjugated to column matrices and the proteins immunopurified.All of these methods are well known in the art. It will be apparent toone of skill that chromatographic techniques can be performed at anyscale and using equipment from many different manufacturers (e.g.,Pharmacia Biotech).

[0157] V. Immunological Detection of p47ING3.

[0158] In addition to the detection of p47ING3 genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect p47ING3, e.g., to identify alleles, mutants,polymorphic variants and interspecies homologs of p47ING3. Immunoassayscan be used to qualitatively or quantitatively analyze p47ING3, e.g., todetect p47ING3, to measure p47ING3 activity, or to identify modulatorsof p47ING3 activity. A general overview of the applicable technology canbe found in Harlow and Lane, Antibodies: A Laboratory Manual (1988).

[0159] A. Antibodies to p47ING3

[0160] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with p47ING3 are known to those of skill in the art(see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow &Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2^(nd) ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975).Such techniques include antibody preparation by selection of antibodiesfrom libraries of recombinant antibodies in phage or similar vectors, aswell as preparation of polyclonal and monoclonal antibodies byimmunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Inaddition, as noted above, many companies, such as BMA Biomedicals, Ltd.,HTI Bio-products, and the like, provide the commercial service of makingan antibody to essentially any peptide.

[0161] A number of p47ING3 comprising immunogens may be used to produceantibodies specifically reactive with p47ING3. For example, recombinantp47ING3, or antigenic fragments thereof, are isolated as describedherein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

[0162] Methods of production of polyclonal antibodies are known to thoseof skill in the art. To improve reproducibility, an inbred strain ofmice (e.g., BALB/C mice) can be immunized to make the antibody; however,standard animals (mice, rabbits, etc.) used to make antibodies areimmunized with the protein using a standard adjuvant, such as Freund'sadjuvant, and a standard immunization protocol (see Harlow & Lane,supra). The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the protein of choice. When appropriately high titers of antibody tothe immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired (seeHarlow & Lane, supra).

[0163] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989).

[0164] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against non-p47ING3proteins or even other related proteins, e.g., from other organisms,using a competitive binding immunoassay. Specific polyclonal antiseraand monoclonal antibodies will usually bind with K_(D) of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

[0165] Once p47ING3 specific antibodies are available, these proteinscan be detected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

[0166] B. Immunological Binding Assays

[0167] p47ING3 can be detected and/or quantified using any of a numberof well recognized immunological binding assays (see, e.g., U.S. Pat.Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review ofthe general immunoassays, see also Methods in Cell Biology: Antibodiesin Cell Biology, volume 37 (Asai, ed. 1993); Basic and ClinicalImmunology (Stites & Terr, eds., 7th ed. 1991). Immunological bindingassays (or immunoassays) typically use an antibody that specificallybinds to a protein or antigen of choice (in this case p47ING3, orantigenic fragments thereof). The antibody may be produced by any of anumber of means well known to those of skill in the art and as describedabove.

[0168] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeledp47ING3 polypeptide or a labeled anti-p47ING3 antibody. Alternatively,the labeling agent may be a third moiety, such a secondary antibody,that specifically binds to the antibody/antigen complex (a secondaryantibody is typically specific to antibodies of the species from whichthe first antibody is derived). Other proteins capable of specificallybinding immunoglobulin constant regions, such as protein A or protein Gmay also be used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

[0169] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0170] Non-Competitive Assay Formats

[0171] Immunoassays for detecting p47ING3 in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-antigen antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture antigen present in the test sample.Antigen thus immobilized is then bound by a labeling agent, such as asecond antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

[0172] Competitive Assay Formats

[0173] In competitive assays, the amount of p47ING3 present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) antigen displaced (competed away) from an anti-antigenantibody by the unknown antigen present in a sample. In one competitiveassay, a known amount of antigen is added to a sample and the sample isthen contacted with an antibody that specifically binds to the antigen.The amount of exogenous antigen bound to the antibody is inverselyproportional to the concentration of antigen present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of antigen bound to the antibody may bedetermined either by measuring the amount of antigen present in anantigen/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of antigen may be detected byproviding a labeled antigen molecule.

[0174] A hapten inhibition assay is another preferred competitive assay.In this assay the known antigen is immobilized on a solid substrate. Aknown amount of anti-antigen antibody is added to the sample, and thesample is then contacted with the immobilized antigen. The amount ofanti-antigen antibody bound to the known immobilized antigen isinversely proportional to the amount of antigen present in the sample.Again, the amount of immobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0175] Cross-Reactivity Determinations

[0176] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations. For example, p47ING3 proteins can beimmobilized to a solid support. Proteins (e.g., p33ING1 or p33ING2) areadded to the assay that compete for binding of the antisera to theimmobilized antigen. The ability of the added protein to compete forbinding of the antisera to the immobilized protein is compared to theability of antigen to compete with itself. The percent cross-reactivityfor the above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with the added proteins areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the addedproteins.

[0177] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein thought to be perhaps an allele, interspecies homologs, orpolymorphic variant of p47ING3, to the immunogen protein. In order tomake this comparison, the two proteins are each assayed at a wide rangeof concentrations and the amount of each protein required to inhibit 50%of the binding of the antisera to the immobilized protein is determined.If the amount of the second protein required to inhibit 50% of bindingis less than 10 times the amount of the first protein that is requiredto inhibit 50% of binding, then the second protein is said tospecifically bind to the polyclonal antibodies generated to theimmunogen of choice.

[0178] Other Assay Formats

[0179] Western blot (immunoblot) analysis is used to detect and quantifythe presence of p47ING3 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind p47ING3. The anti-antigen antibodies specificallybind to the antigen on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-antigen antibodies.

[0180] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:3441 (1986)).

[0181] Reduction of Non-Specific Binding

[0182] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0183] Labels

[0184] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general., most any label useful insuch methods can be applied to the present invention. Thus, a label isany composition detectable by spectroscopic, photochemical.,biochemical., immunochemical., electrical., optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.,DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g. horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0185] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0186] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize a specific protein, or secondary antibodies thatrecognize antibodies to the specific protein.

[0187] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0188] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0189] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0190] VI. Assays for Measuring Changes in p47ING3 Regulated Cell Growth

[0191] p47ING3 and its alleles, interspecies homologs, and polymorphicvariants participate in regulation of cell proliferation and tumorsuppression. Therefore, expression of p47ING3 and its alleles,interspecies homologs, and polymorphic variants in host cells wouldinhibit cell proliferation and suppress tumor formation. On the otherhand, expression of p47ING3 mutants in a cell would lead to abnormalcell proliferation and loss of tumor suppressor phenotypes. Finally,compounds that activate or inhibit p47ING3 would indirectly affectregulation of cellular proliferation and tumor suppression. Any of thesechanges in cell growth can be assessed by using a variety of in vitroand in vivo assays, e.g., ability to grow on soft agar, changes incontact inhibition and density limitation of growth, changes in growthfactor or serum dependence, changes in the level of tumor specificmarkers, changes in invasiveness into Matrigel, changes in tumor growthin vivo, such as in transgenic mice, etc. Furthermore, these assays canbe to screen for activators, inhibitors, and modulators of p47ING3. Suchactivators, inhibitors, and modulators of p47ING3 can then be used tomodulate p47ING3 expression in tumor cells or abnormal proliferativecells.

[0192] A. Assays for Changes in Cell Growth by Expression of p47ING3Constructs

[0193] The following are assays that can be used to identify p47ING3constructs which are capable of regulating cell proliferation and tumorsuppression. The phrase “p47ING3 constructs” can refer to any of p47ING3and its alleles, interspecies homologs, polymorphic variants andmutants. Functional p47ING3 constructs identified by the followingassays can then be used in gene therapy to inhibit abnormal cellularproliferation and transformation.

[0194] Soft Agar Growth or Colony Formation in Suspension

[0195] Normal cells require a solid substrate to attach and grow. Whenthe cells are transformed, they lose this phenotype and grow detachedfrom the substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with tumorsuppressor genes, regenerate normal phenotype and require a solidsubstrate to attach and grow.

[0196] Soft agar growth or colony formation in suspension assays can beused to identify p47ING3 constructs, which when expressed in host cells,inhibit abnormal cellular proliferation and transformation. Typically,transformed host cells (e.g. cells that grow on soft agar) are used inthis assay. Expression of a tumor suppressor gene in these transformedhost cells would reduce or eliminate the host cells' ability to grow instirred suspension culture or suspended in semi-solid media, such assemi-solid or soft. This is because the host cells would regenerateanchorage dependence of normal cells, and therefore require a solidsubstrate to grow. Therefore, this assay can be used to identify p47ING3constructs which function as a tumor suppressor. Once identified, suchp47ING3 constructs can be used in gene therapy to inhibit abnormalcellular proliferation and transformation.

[0197] Techniques for soft agar growth or colony formation in suspensionassays are described in Freshney, Culture of Animal Cells a Manual ofBasic Technique, 3^(rd) ed., Wiley-Liss, New York (1994), hereinincorporated by reference. See also, the methods section of Garkavtsevet al. (1996), supra, herein incorporated by reference.

[0198] Contact Inhibition and Density Limitation of Growth

[0199] Normal cells typically grow in a flat and organized pattern in apetri dish until they touch other cells. When the cells touch oneanother, they are contact inhibited and stop growing. When cells aretransformed, however, the cells are not contact inhibited and continueto grow to high densities in disorganized foci. Thus, the transformedcell grow to a higher saturation density than normal cells. This can bedetected morphologically by the formation of a disoriented monolayer ofcells or rounded cells in foci within the regular pattern of normalsurrounding cells. Alternatively, labeling index with [³H]-thymidine atsaturation density can be used to measure density limitation of growth.See Freshney (1994), supra. The transformed cells, when transfected withtumor suppressor genes, regenerate a normal phenotype and become contactinhibited and would grow to a lower density.

[0200] Contact inhibition and density limitation of growth assays can beused to identify p47ING3 constructs which are capable of inhibitingabnormal proliferation and transformation in host cells. Typically,transformed host cells (e.g. cells that are not contact inhibited) areused in this assay. Expression of a tumor suppressor gene in thesetransformed host cells would result in cells which are contact inhibitedand grow to a lower saturation density than the transformed cells.Therefore, this assay can be used to identify p47ING3 constructs whichfunction as a tumor suppressor. Once identified, such p47ING3 constructscan be used in gene therapy to inhibit abnormal cellular proliferationand transformation.

[0201] In this assay, labeling index with [³H]-thymidine at saturationdensity is a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a p47ING3 construct and aregrown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with [³H]-thymidine isdetermined autoradiographically. See, Freshney (1994), supra. The hostcells expressing a functional p47ING3 construct would give arise to alower labeling index compared to control (e.g., transformed host cellstransfected with a vector lacking an insert).

[0202] Growth Factor or Serum Dependence

[0203] Growth factor or serum dependence can be used as an assay toidentify functional p47ING3 constructs. Transformed cells have a lowerserum dependence than their normal counterparts (see, e.g., Temin, J.Natl. Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med.131:836-879 (1970)); Freshney, supra. This is in part due to release ofvarious growth factors by the transformed cells. When a tumor suppressorgene is transfected and expressed in these transformed cells, the cellswould reacquire serum dependence and would release growth factors at alower level. Therefore, this assay can be used to identify p47ING3constructs which function as a tumor suppressor. Growth factor or serumdependence of transformed host cells which are transfected with ap47ING3 construct can be compared with that of control (e.g.,transformed host cells which are transfected with a vector withoutinsert). Host cells expressing a functional p47ING3 would exhibit anincrease in growth factor and serum dependence compared to control.

[0204] Tumor Specific Markers Levels

[0205] Tumor cells release an increased amount of certain factors(hereinafter “tumor specific markers”) than their normal counterparts.For example, plasminogen activator (PA) is released from human glioma ata higher level than from normal brain cells (see, e.g., Gullino,Angiogenesis, tumor vascularization, and potential interference withtumor growth. In Mihich (ed.): “Biological Responses in Cancer.” NewYork, Academic Press, pp. 178-184 (1985)). Similarly, Tumor angiogenesisfactor (TAF) is released at a higher level in tumor cells than theirnormal counterparts. See, e.g., Folkman, Angiogenesis and cancer, SemCancer Biol. (1992)).

[0206] Tumor specific markers can be assayed for to identify p47ING3constructs, which when expressed, decrease the level of release of thesemarkers from host cells. Typically, transformed or tumorigenic hostcells are used. Expression of a tumor suppressor gene in these hostcells would reduce or eliminate the release of tumor specific markersfrom these cells. Therefore, this assay can be used to identify p47ING3constructs which function as a tumor suppressor.

[0207] Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980);Gulino, Angiogenesis, tumor vascularization, and potential interferencewith tumor growth. In Mihich, E. (ed): “Biological Responses in Cancer.”New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).

[0208] Invasiveness into Matrigel

[0209] The degree of invasiveness into Matrigel or some otherextracellular matrix constituent can be used as an assay to identifyp47ING3 constructs which are capable of inhibiting abnormal cellproliferation and tumor growth. Tumor cells exhibit a good correlationbetween malignancy and invasiveness of cells into Matrigel or some otherextracellular matrix constituent. In this assay, tumorigenic cells aretypically used as host cells. Expression of a tumor suppressor gene inthese host cells would decrease invasiveness of the host cells.Therefore, functional p47ING3 constructs can be identified by measuringchanges in the level of invasiveness between the host cells before andafter the introduction of p47ING3 constructs. If a p47ING3 constructfunctions as a tumor suppressor, its expression in tumorigenic hostcells would decrease invasiveness.

[0210] Techniques described in Freshney (1994), supra, can be used.Briefly, the level of invasion of host cells can be measured by usingfilters coated with Matrigel or some other extracellular matrixconstituent. Penetration into the gel, or through to the distal side ofthe filter, is rated as invasiveness, and rated histologically by numberof cells and distance moved, or by prelabeling the cells with ¹²⁵I andcounting the radioactivity on the distal side of the filter or bottom ofthe dish. See, e.g., Freshney (1984), supra.

[0211] Cell Cycle Analysis

[0212] Cell cycle analysis can be used to determine if a gene cansuppress the growth of a cell. Briefly, cells are transfected with anexpression cassette containing the gene of interest. If the gene encodesa protein that can arrest or inhibit cell division then the gene issuppressing the growth of the cells. Cell division, or mitosis, consistsof several successive phases in a eukaryotic cell (Molecular Biology ofthe Cell, 3d edition (Alberts et al., eds., 1994)). These phases, inorder, are known as G₁, S, G₂ and M. DNA replication takes place duringthe S phase. The mitotic phase, where nuclear division takes place, istermed the M phase. The G₁ phase is the time between the M phase and theS phase. G₂ is the time between the end of the S phase and the beginningof the M phase. Cells can pause in G₁ and enter a specialized restingstate known as G₀. Cells can remain in G₀ for days to years, until theyresume the cell-cycle. Methods of analyzing the phase of the cell-cycleare known in the art and include methods that involve determining if thecell is replicating DNA (e.g., [³H]-thymidine incorporation assays).Alternatively, methods are known in the art for measuring the DNAcontent of a cell, which doubles during the S phase. FACS (Fluorescentactivated cell sorting) analysis can be used to determine the percentageof a population of cells in a particular stage of the cell-cycle (seegenerally, Alberts et al., supra; see also van den Heuvel and Harlow,(1993) Science 262: 2050-2054). The cells are incubated with a dye thatfluoresces (e.g., propidium iodide) when it binds to the DNA of thecell. Thus, the amount of fluorescence of a cell is proportional to theDNA content of a cell. Cells that are in G₁ or G₀ (G₁/G₀) have an

replicated complement of DNA and are deemed to have 1 arbitrary unit ofDNA in the cell. Those cells that have fully replicated, i.e., havedoubled their DNA content, are deemed to have 2 arbitrary units of DNAin the cell and are in the G₂ or M phase (G₂/M) of the cell cycle. Cellswith an amount of DNA that is between 1 and 2 arbitrary units are in Sphase.

[0213] The effect of a protein of interest on the cell cycle can bedetermined by transfecting cells with DNA encoding the protein ofinterest and analyzing its effect on the cell cycle through flowcytometry in a FACS. The cells are co-transfected with a vector encodinga marker to identify and analyze those cells that are actuallytransfected. Such markers can include the B cell surface marker CD20(van de Heuvel and Harlow, supra) or a farnesylated green fluorescentprotein (GFP-F) (Jiang and Hunter, (1998) Biotechniques, 24(3): 349-50,352, 354).

[0214] For example, the percentage of cells in a particular stage of thecell-cycle can be determined using the method of Jiang and Hunter,(1998) supra. Briefly, a population of cells are transfected with avector encoding p47ING3 and a vector encoding a green fluorescentprotein (GFP) with a famesylation signal sequence from c-Ha-Ras. Thefamesylation signal sequence is farnesylated in the cell, which targetsthe GFP molecule to the plasma membrane. Vectors encoding farnesylatedGFP are commercially available (e.g., pEGFP-F from Clontech).

[0215] After transfection, the cells are suspended in buffer containingthe DNA intercalator propidium iodide. Propidium iodide will fluorescewhen it is bound to DNA. Thus, the amount of fluorescence observed frompropidium iodide in a FACS flow cytometer is an indication of the DNAcontent of a cell. The percentages of cells in each cell cycle can becalculated using computer programs, e.g., the ModFit program(Becton-Dickinson). The cell cycle stage of the cell was analyzed aftergating cells by GFP fluorescence using FACscan. If the gene encodes atumor suppressor, the percentage of cells that enter S phase would bedecreased, as the cells are arrested in the G₀/G₁ phase. Therefore, thepercentage of cells that are G₀/G₁ phase would be increased.

[0216] Tumor Growth In Vivo

[0217] Effects of p47ING3 on cell growth can be tested in transgenic orimmune-suppressed mice. Knock-out transgenic mice can be made, in whichthe endogenous p47ING3 gene is disrupted. Such knock-out mice can beused to study effects of p47ING3, e.g., as a cancer model, as a means ofassaying in vivo for compounds that modulate p47ING3, and to test theeffects of restoring a wildtype p47ING3 to a knock-out mice.

[0218] Knock-out transgenic mice can be made by insertion of a markergene or other heterolgous gene into the endogenous p47ING3 gene site inthe mouse genome via homologous recombination. Such mice can also bemade by substituting the endogenous p47ING3 with a mutated version ofp47ING3, or by mutating the endogenous p47ING3, e.g., by exposure tocarcinogens.

[0219] A DNA construct is introduced into the nuclei of embryonic stemcells. Cells containing the newly engineered genetic lesion are injectedinto a host mouse embryo, which is re-implanted into a recipient female.Some of these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual., Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

[0220] These knock-out mice can be used as hosts to test the effects ofvarious p47ING3 constructs on cell growth. These transgenic mice withthe endogenous p47ING3 gene knocked out would develop abnormal cellproliferation and tumor growth. They can be used as hosts to test theeffects of various p47ING3 constructs on cell growth. For example,introduction of wildtype p47ING3 into these knock-out mice would inhibitabnormal cellular proliferation and suppress tumor growth.

[0221] Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, genetically athymic “nude” mouse (see,e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCIDmouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradleyet al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52(1980)) can be used as a host. Transplantable-tumor cells (typicallyabout 10⁶ cells) injected into isogenic hosts will produce invasivetumors in a high proportions of cases, while normal cells of similarorigin will not. In hosts which developed invasive tumors, cellsexpressing a p47ING3 construct are injected subcutaneously. After asuitable length of time, preferably 4-8 weeks, tumor growth is measured(e.g., by volume or by its two largest dimensions) and compared to thecontrol. Tumors that have statistically significant reduction (using,e.g., Student's T test) are said to have inhibited growth. Usingreduction of tumor size as an assay, functional p47ING3 constructs whichare capable of inhibiting abnormal cell proliferation can be identified.This model can also be used to identify mutant versions of p47ING3.

[0222] B. Assays for Compounds that Modulate p47ING3

[0223] p47ING3 and its alleles, interspecies homologs, and polymorphicvariants participate in regulation of cell proliferation and tumorsuppression. Mutations in these genes, including null or missensemutations, can cause abnormal cell proliferation and tumor growth. Theactivity of p47ING3 polypeptides (wildtype or mutants) can be assessedusing a variety of in vitro and in vivo assays measuring variousparameters, e.g., cell growth on soft agar, contact inhibition anddensity limitation of growth, growth factor or serum dependence, tumorspecific markers levels, invasiveness into Matrigel, tumor growth invivo, transgenic mice, p47ING3 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene, and the like. Such assayscan also be used to screen for activators, inhibitors, and modulators ofwildtype and mutant p47ING3. Such activators, inhibitors, and modulatorsare useful in inhibiting tumor growth and modulating cell proliferation.Compounds identified using the assays of the invention are useful astherapeutics for treatment of cancer and other diseases involvingcellular hyperproliferation.

[0224] Biologically active or inactivated p47ING3 polypeptides, eitherrecombinants or naturally occurring, are used to screen activators,inhibitors, or modulators of tumor suppression and cell proliferation.The p47ING3 polypeptides can be recombinantly expressed in a cell,naturally expressed in a cell, recombinantly or naturally expressed incells transplanted into an animal., or recombinantly or naturallyexpressed in a transgenic animal. Modulation is tested using one of thein vitro or in vivo assays described in herein in part A.

[0225] Cells that have wildtype p47ING3 are used in the assays of theinvention, both in vitro and in vivo. Preferably, human cells are used.Cell lines can also be created or isolated from tumors that have mutantp47ING3. Optionally, the cells can be transfected with an exogenousp47ING3 gene operably linked to a constitutive promoter, to providehigher levels of p47ING3 expression. Alternatively, endogenous p47ING3levels can be examined. The cells can be treated to induce p47ING3expression. The cells can be immobilized, be in solution, be injectedinto an animal., or be naturally occurring in a transgenic ornon-transgenic animal.

[0226] Samples or assays that are treated with a test compound whichpotentially activates, inhibits, or modulates p47ING3 are compared tocontrol samples that are not treated with the test compound, to examinethe extent of modulation. Generally, the compounds to be tested arepresent in the range from 0.1 nM to 10 mM. Control samples (untreatedwith activators, inhibitors, or modulators) are assigned relativep47ING3 activity value of 100%. Inhibition of p47ING3 is achieved whenthe p47ING3 activity value relative to the control is about 90% (e.g.,10% less than the control), preferably 50%, more preferably 25%.Activation of p47ING3 is achieved when the p47ING3 activity valuerelative to the control is 110% (e.g., 10% more than the control), morepreferably 150%, more preferably 200% higher.

[0227] The effects of the test compounds upon the function of thep47ING3 polypeptides can be measured by examining any of the parametersdescribed above. For example, parameters such as ability to grow on softagar, contact inhibition and density limitation of growth, growth factoror serum dependence, tumor specific markers levels, invasiveness intoMatrigel, tumor growth in vivo, transgenic mice and the like, can bemeasured. Furthermore, the effects of the test compounds on p47ING3protein or mRNA levels, transcriptional activation or repression of areporter gene can be measured. In each assay, cells expressing p47ING3are contacted with a test compound and incubated for a suitable amountof time, e.g., from 0.5 to 48 hours. Then, parameters such as thosedescribed above are compared to those produced by control cellsuntreated with the test compound.

[0228] In one embodiment, the effect of test compounds upon the functionof p47ING3 can be determined by comparing the level of p47ING3 proteinor mRNA in treated samples and control samples. The level of p47ING3protein is measured using immunoassays such as western blotting, ELISAand the like with a p47ING3 specific antibody. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,northern hybridization, RNase protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

[0229] Alternatively, a reporter gene system can be devised using thep47ING3 promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or β-gal. After treatment with apotential p47ING3 modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

[0230] In another embodiment, the effects of test compounds on p47ING3activity is performed in vivo. In this assay, cultured cells that areexpressing a wildtype or mutant p47ING3 (e.g., a null or missensemutation) are injected subcutaneously into an immune compromised mousesuch as an athymic mouse, an irradiated mouse, or a SCID mouse. Thep47ING3 modulators are administered to the mouse, e.g., a chemicalligand library. After a suitable length of time, preferably 4-8 weeks,tumor growth is measured, e.g. by volume or by its two largestdimensions, and compared to the control. Tumors that have statisticallysignificant reduction (using, e.g., Student's T test) are said to haveinhibited growth. Alternatively, the extent of tumor neovascularizationcan also be measured. Immunoassays using endothelial cell specificantibodies are used to stain for vascularization of the tumor and thenumber of vessels in the tumor. Tumors that have a statisticallysignificant reduction in the number of vessels (using, e.g., Student's Ttest) are said to have inhibited neovascularization.

[0231] Alternatively, transgenic mice with the endogenous p47ING3 geneknocked out can be used in an assay to screen for compounds whichmodulate the p47ING3 activity. As described in part A, knock-outtransgenic mice can be made, in which the endogenous p47ING3 gene isdisrupted, e.g., by replacing it with a marker gene. A transgenic mousethat is heterozygous or homozygous for integrated transgenes that havefunctionally disrupted the endogenous p47ING3 gene can be used as asensitive in vivo screening assay for p47ING3 ligands and modulators ofp47ING3 activity.

[0232] C. Modulators

[0233] The compounds tested as modulators of p47ING3 can be any smallchemical compound, or a biological entity, such as a protein, sugar,nucleic acid or lipid. Alternatively, modulators can be geneticallyaltered versions of p47ING3. For example, a n antisense construct ofp47ING3 can be used as a modulator.

[0234] Typically, test compounds will be small chemical molecules andpeptides. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although most oftencompounds can be dissolved in aqueous or organic (especially DMSO-based)solutions are used. The assays are designed to screen large chemicallibraries by automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). It willbe appreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

[0235] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0236] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0237] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (19

)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658(1994)), nucleic acid libraries (see Ausubel et al., supra, Berger andSambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S.Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries(see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, 5,288,514, and the like).

[0238] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0239] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell or tissueexpressing p47ING3 is attached to a solid phase substrate. In the highthroughput assays of the invention, it is possible to screen up toseveral thousand different modulators or ligands in a single day. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped, e.g., by Caliper Technologies (Palo Alto, Calif.).

[0240] D. Computer-Based Assays

[0241] Yet another assay for compounds that modulate p47ING3 activityinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of p47ING3 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

[0242] The three-dimensional structural model of the protein isgenerated by entering p47ING3 amino acid sequences of at least 10 aminoacid residues or corresponding nucleic acid sequences encoding a p47ING3polypeptide into the computer system. The amino acid sequence of thepolypeptide or the nucleic acid encoding the polypeptide is selectedfrom the group consisting of SEQ ID NO:1 or SEQ ID NO:2, andconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence or subsequence of the protein, whichencodes the structural information of the protein. At least 10 residuesof the amino acid sequence (or a nucleotide sequence encoding 10 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe protein is then generated by the interaction of the amino acidsequence and the computer system, using software known to those of skillin the art. The three-dimensional structural model of the protein can besaved to a computer readable form and be used for further analysis(e.g., identifying potential ligand binding regions of the protein andscreening for mutations, alleles and interspecies homologs of the gene).

[0243] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the protein of interest. The software looks atcertain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as-“energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

[0244] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0245] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of the p47ING3 protein to identify ligands that bind top47ING3. Binding affinity between the protein and ligands is determinedusing energy terms to determine which ligands have an enhancedprobability of binding to the protein. The results, such asthree-dimensional structures for potential ligands and binding affinityof ligands, can also be saved to a computer readable form and can beused for further analysis (e.g., generating a three dimensional model ofmutated proteins having an altered binding affinity for a ligand).

[0246] Computer systems are also used to screen for mutations,polymorphic variants, alleles and interspecies homologs of p47ING3genes. Such mutations can be associated with disease states or genetictraits. As described above, high density oligonucleotide arrays(GeneChip™) and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutatedp47ING3 genes involves receiving input of a first nucleic acid or aminoacid sequence encoding selected from the group consisting of SEQ IDNO:2, or SEQ ID NO:1, and conservatively modified versions thereof. Thesequence is entered into the computer system as described above and thensaved to a computer readable form. The first nucleic acid or amino acidsequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in p47ING3 genes, androtations associated with disease states and genetic traits.

[0247] VII. Gene Therapy

[0248] The present invention provides the nucleic acids of p47ING3 forthe transfection of cells in vitro and in vivo. These nucleic acids canbe inserted into any of a number of well known vectors for thetransfection of target cells and organisms as described below. Thenucleic acids are transfected into cells, ex vivo or in vivo, throughthe interaction of the vector and the target cell. The nucleic acidsencoding p47ING3, under the control of a promoter, then expresses ap47ING3 of the present invention, thereby mitigating the effects ofabsent, partial inactivation, or abnormal expression of the p47ING3gene.

[0249] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies (for a review of gene therapy procedures, see Anderson,Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):3144 (1995); Haddada et al.,in Current Topics in Microbiology and Immunology (Doerfler & Böhm eds.,1995); and Yu et al., Gene Therapy 1:13-26 (1994)).

[0250] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):3144 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

[0251] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (exvivo administration) or target tissues (in vivo administration).

[0252] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to oneof skill in the art (see, e.g., Crystal., Science 270:404-410 (1995);Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad etal., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,4,837,028, and 4,946,787).

[0253] The use of RNA or DNA viral based systems for the delivery ofnucleic acids take advantage of highly evolved processes for targeting avirus to specific cells in the body and trafficking the viral payload tothe nucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo) Conventional viral based-systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

[0254] The tropism of a retrovirus can be altered by incorporatingforeign envelope proteins, expanding the potential target population oftarget cells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

[0255] In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

[0256] In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

[0257] pLASN and MFG-S are examples are retroviral vectors that havebeen used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995);Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl.Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the firsttherapeutic vector used in a gene therapy trial. (Blaese et al., Science270:475-480 (1995)). Transduction efficiencies of 50% or greater havebeen observed for MFG-S packaged vectors. (Ellem et al., ImmunolImmunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111′-2(1997).

[0258] Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

[0259] Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

[0260] Packaging cells are used to form virus particles that are capableof infecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g. heattreatment to which adenovirus is more sensitive than AAV.

[0261] In many gene therapy applications, it is desirable that the genetherapy vector be delivered with a high degree of specificity to aparticular tissue type. A viral vector is typically modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the viruses outer surface. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. U.S.A. 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g. Fab or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0262] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal., intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

[0263] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith a nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

[0264] In one embodiment, stem cells are used in ex vivo procedures forcell transfection and gene therapy. The advantage to using stem cells isthat they can be differentiated into other cell types in vitro, or canbe introduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

[0265] Stem cells are isolated for transduction and differentiationusing known methods. For example, stem cells are isolated from bonemarrow cells by panning the bone marrow cells with antibodies which bindunwanted cells, such as CD4+ and CD8+ (T cells), CD45+ (panB cells),GR-1 (granulocytes), and Iad (differentiated antigen presenting cells)(see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

[0266] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be also administered directlyto the organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered.

[0267] Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cells,as described below. The nucleic acids are administered in any suitablemanner, preferably with pharmaceutically acceptable carriers. Suitablemethods of administering such nucleic acids are available and well knownto those of skill in the art, and, although more than one route can beused to administer a particular composition, a particular route canoften provide a more immediate and more effective reaction than anotherroute (see Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989)). In particular, atleast six viral vector approaches are currently available for genetransfer in clinical trials, with retroviral vectors by far the mostfrequently used system. All of these viral vectors utilize approachesthat involve complementation of defective vectors by genes inserted intohelper cell lines to generate the transducing agent.

[0268] VIII. Pharmaceutical Compositions and Administration

[0269] p47ING3 nucleic acid, protein, and modulators of p47ING3 can beadministered directly to the patient for inhibition of cancer, tumor, orprecancer cells in vivo. Administration is by any of the routes normallyused for introducing a compound into ultimate contact with the tissue tobe treated. The compounds are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such compounds are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

[0270] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985)).

[0271] The compounds (nucleic acids, proteins, and modulators), alone orin combination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

[0272] Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal., and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

[0273] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular compound employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular compound or vector in a particularpatient.

[0274] In determining the effective amount of the modulator to beadministered in the treatment or prophylaxis of cancer, the physicianevaluates circulating plasma levels of the modulator, modulatortoxicities, progression of the disease, and the production ofanti-modulator antibodies. In general., the dose equivalent of amodulator is from about 1 ng/kg to 10 mg/kg for a typical patient.Administration of compounds is well known to those of skill in the art(see, e.g., Bansinath et al., Neurochem Res. 18:1063-1066 (1993);Iwasaki et al., Jpn. J. Cancer Res. 88:861-866 (1997); Tabrizi-Rad etal., Br. J. Pharmacol. 111:394-396(1994)).

[0275] For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

[0276] IX. Diagnostics and Kits

[0277] The present invention also provides methods for detection ofp47ING3 (either wildtype or mutant). For example, kits are provided thatcontain p47ING3 specific reagents that specifically hybridize to p47ING3nucleic acid, such as specific probes and primers, and p47ING3 specificreagents that specifically bind to the protein of choice, e.g.,antibodies. The methods, kits, and the assays described herein can beused for identification of modulators of p47ING3, or for diagnosingpatients with mutations in p47ING3.

[0278] Nucleic acid assays for the presence of p47ING3 DNA and RNA in asample include numerous techniques are known to those skilled in theart. In particular, p47ING3 specific reagents (e.g., p47ING3-specificprimers or nucleic acid probes) can be used to distinguish betweensamples which contain p47ING3 nucleic acids and samples which containp33ING1 or p33ING2 nucleic acids. Techniques such as Southern analysis,Northern analysis, dot blots, RNase protection, high densityoligonucleotide arrays, S1 analysis, amplification techniques such asPCR and LCR, and in situ hybridization can be used as assays. In in situhybridization, for example, the target nucleic acid is liberated fromits cellular surroundings in such as to be available for hybridizationwithin the cell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987).

[0279] In addition, p47ING3 protein can be detected with the variousimmunoassay techniques described above, e.g., ELISA, Western blotting,and the like. The test sample is typically compared to both a positivecontrol (e.g., a sample expressing recombinant p47ING3) and a negativecontrol. In particular, p47ING3, p33ING1 or p33ING2 specific polyclonaland monoclonal antibodies or specific polyclonal and monoclonalantibodies can be used as a diagnostic tool to distinguish betweensamples which contain p47ING3, p33ING1, or p33ING2 antigens.

[0280] The present invention also provides for kits for screening formodulators of p47ING3. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: p47ING3, reaction tubes, andinstructions for testing p47ING3 activity. Preferably, the kit containsbiologically active p47ING3. A wide variety of kits and components canbe prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

EXAMPLES

[0281] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

Example I Cloning and Expression of p47ING3

[0282] p47ING3 homologous sequences were found in a random cDNA sequencedatabase consisting of short partial sequences known as expressedsequence tags (ESTs) submitted in GenBank. Using primers designed basedon these EST sequences and using RT-PCR and 5′- and 3′-RACE methods,p47ING3 coding region (SEQ ID NO:2) from human placenta cDNA (CLONTECH)was isolated and subcloned into a plasmid. The amino acid sequence ofp47ING3 (SEQ ID NO: 1) has about 34% amino acid identity with p33ING1(SEQ ID NO: 8 (p33ING1)) and p33ING2 (SEQ ID NO: 6 (p33ING2).

Example II Antibodies to p47ING3 Against Interior Peptide Sequence

[0283] Antibodies to p47ING3 were synthesized against the peptide ping-3from p47ING3 (SEQ ID NO:5: HTPVEKRKYNPTSHHTT). The peptide is aminoacids 141-157 of SEQ ID NO: 1. The peptide was purified by HPLC; peptideKLH conjugations were made; and rabbits were immunized by them.Antiserum was purified using peptide affinity column and specificity ofeach polyclonal antibody was analyzed by ELISA.

[0284] p33ING1, p33ING2, and p47ING3 proteins were produced by Promega'sTNT Quick Coupled Transcription/Translation System (Rabbit ReticulocyteLysate) from pcDNA3.1-ING1, ING2, and ING3 expression vectors.

[0285] Plasmids encoding p33ING1, p33ING2, and p47ING3 were separatelysubjected to an in vitro transcription/translation system to produce therespective proteins. The proteins were electrophoresed on SDS-PAGE andWestern blotted. The blot was incubated with anti-p47ING3 polyclonalantibodies and detection was performed using a horse radish peroxidaselabelled system from Amersham Pharmacia Biotech. As shown in FIG. 1,anti-p47ING3 polyclonal antibodies are reactive with recombinant p47ING3protein, but are not cross-reactive with recombinant p33ING1 protein orrecombinant p33ING2 protein. The p47ING3 protein migrates an a sizeappropriate for its predicted molecular weight of 47 kDa.

Example III Antibodies to p47ING3 Against N-Terminal Sequence

[0286] Antibodies to p47ING3 were synthesized against the peptidePing-3N from p47ING3 (SEQ ID NO:9: MLYLEDYLEM; amino acids 1-10 of SEQID NO: 1). The peptide was purified by HPLC; peptide KLH conjugationswere made; and rabbits were immunized by them. Antiserum was purifiedusing peptide affinity column and specificity of each polyclonalantibody was analyzed by ELISA and Western blot analysis.

[0287] p33ING1, p33ING2, and p47ING3 were produced by Promega's TNTQuick Coupled Transcription/Translation System (Rabbit ReticulocyteLysate) from pcDNA3.1-ING1, ING2, and ING3 expression vectors.

[0288] Plasmids encoding p33ING1, p33ING2, and p47ING3 were separatelysubjected to an in vitro transcription/translation system to produce therespective proteins. The proteins were electrophoresed on SDS-PAGE withmolecular weight markers (Kaleidoscope Pre

ained Standards; Bio-Rad; 161-0324) and Western blotted to a Immobilon-Pmembrane (Millipore, IPVH15150). The blot was incubated withanti-p47ING3 polyclonal antibodies (1:200 dilution) and detection wasperformed using a secondary antibody—goat anti-rabbit IgG HRP conjugate(Santa Cruz Biotechnology, sc-2004) (1:2000 dilution). Detection wascarried out using ECL Western Blotting Detection Reagents (AmershamPharmacia Biotech, RPN2106) and Hyperfilm ECL (Amersham PharmaciaBiotech, RPN2103K). The autoradiogram of the Western blot is depicted asFIG. 2.

[0289] As shown in FIG. 2, anti-p47ING3 polyclonal antibodies arereactive with recombinant p47ING3 protein, but are not cross-reactivewith recombinant p33ING1 protein or recombinant p33ING2 protein. Theimmunoreactive band in the p47ING3 lane protein migrates an a sizeappropriate for the predicted molecular weight of p47ING3 of 47 kDa.

Example IV Inhibition of Cell Proliferation

[0290] The colony formation assay was used to determine if p47ING3inhibits cell growth of the RKO human colon carcinoma cell line which isavailable from the ATCC. A mammalian expression vector (with CMVpromoter, Neomycin resistant) containing p47ING3 in the senseorientation (pcDNA3.1-p47ING3) was constructed in the expression vectorpcDNA3.1 (Invitrogen). RKO cell lines were transfected with the parentvector pcDNA3.1 or with pcDNA3.1-p47ING3. The transfected cells wereselected with G418 to clone those cells with neomycin resistance. TheRKO (pcDNA3.1) and the RKO (pcDNA3.1-p47ING3) cell lines were subjectedto the colony formation assay was analyze the effect of p47ING3 oncellular proliferation. As shown in FIG. 3, RKO cells transfected withpcDNA3.1-p47ING3 formed less colonies compared to RKO cells transfectedwith pcDNA3.1. This result illustrates that p47ING3 can inhibit cellgrowth.

Example V Cell Cycle Assay of p47ING3 Transfected Cells

[0291] Cell-cycle stage analysis of cells transfected with an expressionvector encoding p47ING3 was performed using the method of Jiang andHunter, supra. The method permits the analysis of cell-cycle profiles intransfected cells using a membrane-targeted green fluorescent protein(GFP).

[0292] RKO cells were plated in a 5(

cm² dish at a density of 1×10⁴ cells/cm². Plasmid DNA mixtures thatcontained 0.15 picomoles of pEGFP-F Amp with either 0.015 picomoles ofpcDNA3.1 (Invitrogen) or 0.015 picomoles of pcDNA3.1-p47ING3 weretransfected using LipofectAMINE reagent (Life Technologies). VectorpEGFP-F Amp (ampicilin resistant) was constructed from vector frompeGFP-F by replacing the kanamycin resistance gene with an ampicilinresistance gene. Vector pEGFP-F is available for Clontech and iskanamycin resistant.

[0293] After 3 hours of incubation, the medium was changed to fresh DMEMwith 10% FBS. After an additional 48 hour incubation, the cells wereharvested, fixed in 70% ethanol and then suspended in PBS containing 20μg/ml propidium iodide and 100 μg/ml RNaseA. The propidium iodide signalwas used as a measure for DNA content to determine cell-cycle profileson a FACScan flow cytometer (Becton-Dickinson). The cell cycle stage ofthe cell was analyzed after gating cells by GFP fluorescence usingFACscan. Cells with a green fluorescent signal at least 2 times strongerthan that in the negative cells are considered GFP-positive and cellswith a signal equal to or less than negatives cells are consideredGFP-negative. Those cells exhibiting a green fluorescent signal at least2 times stronger than that in the negative cells were consideredGFP-positive and cells with a signal equal to or less than negativescells were considered GFP-negative. The percentages of the cells in eachcell cycle phase ((G₀/G₁), S and (G₂/M)) were calculated by the ModFitprogram (Becton-Dickinson) (FIG. 4). The cell-cycle profiles ofGFP-positive and GFP-negative populations from the same dish can becompared. Typically, each DNA histogram contains data from at least10,000 cells. In this experiment, the RKO cells transfected withpcDNA3.1-p47ING3 have 13.4% more cells in the G₀/G₁ phase as compared toRKO cells transfected with pcDNA3.1 (FIG. 4). The percentage of RKO(pcDNA3.1) cells in the S and G₂/M phases is higher than RKO(pcDNA3.1-p47ING3) cells. This indicates that p47ING3 is able toincrease the number of cells in the G₀/G₁ phase. Therefore, it appearsthat p47ING3 is able to induce cell cycle arrest at G₀/G₁ phase anddecrease the number of RKO cells that are entering S phase (DNAsynthesis).

Example VI Soft Agar Assay for Identifying Compounds that Modulatep47ING3

[0294] Wildtype or mutant p47ING3 is expressed in host cells to screencompounds that modulate anchorage dependence of host cells expressingp47ING3. This is achieved by using the method disclosed in Garkavtsev etal. (1996), supra, herein incorporated by reference. NMuMG cells aretransfected with retrovirus produced from a vector containing p47ING3 insense or antisense orientation, or a vector lacking insert (control).The soft agar culture is comprised of two layers: an underlay (DMEM, 10%FCS, 0.6% agar) and an overlay (DMEM, 10% FCS, 0.3% agar), 5×10⁴ cellsare plated in soft agar in 10 cm plates are left at 37° C. for 6-7 weeksbefore being counted. The cells are incubated with a test compound for asuitable amount of time, e.g., for 0.5 to 48 hours, before countingcells. The amount of cells in the test sample is then compared tocontrol cells untreated with the compound.

[0295] All publications, patents, and patent applications cited hereinare hereby incorporated by reference in their entirety for all purposes.

1 9 1 418 PRT Homo sapiens p47ING3 1 Met Leu Tyr Leu Glu Asp Tyr Leu GluMet Ile Glu Gln Leu Pro Met 1 5 10 15 Asp Leu Arg Asp Arg Phe Thr GluMet Arg Glu Met Asp Leu Gln Val 20 25 30 Gln Asn Ala Met Asp Gln Leu GluGln Arg Val Ser Glu Phe Phe Met 35 40 45 Asn Ala Lys Lys Asn Lys Pro GluTrp Arg Glu Glu Gln Met Ala Ser 50 55 60 Ile Lys Lys Asp Tyr Tyr Lys AlaLeu Glu Asp Ala Asp Glu Lys Val 65 70 75 80 Gln Leu Ala Asn Gln Ile TyrAsp Leu Val Asp Arg His Leu Arg Lys 85 90 95 Leu Asp Gln Glu Leu Ala LysPhe Lys Met Glu Leu Glu Ala Asp Asn 100 105 110 Ala Gly Ile Thr Glu IleLeu Glu Arg Arg Ser Leu Glu Leu Asp Thr 115 120 125 Pro Ser Gln Pro ValAsn Asn His His Ala His Ser His Thr Pro Val 130 135 140 Glu Lys Arg LysTyr Asn Pro Thr Ser His His Thr Thr Thr Asp His 145 150 155 160 Ile ProGlu Lys Lys Phe Lys Ser Glu Ala Leu Leu Ser Thr Leu Thr 165 170 175 SerAsp Ala Ser Lys Glu Asn Thr Leu Gly Cys Arg Asn Asn Asn Ser 180 185 190Thr Ala Ser Ser Asn Asn Ala Tyr Asn Val Asn Ser Ser Gln Pro Leu 195 200205 Gly Ser Tyr Asn Ile Gly Ser Leu Ser Ser Gly Thr Gly Ala Gly Ala 210215 220 Ile Thr Met Ala Ala Ala Gln Ala Val Gln Ala Thr Ala Gln Met Lys225 230 235 240 Glu Gly Arg Arg Thr Ser Ser Leu Lys Ala Ser Tyr Glu AlaPhe Lys 245 250 255 Asn Asn Asp Phe Gln Leu Gly Lys Glu Phe Ser Met AlaArg Glu Thr 260 265 270 Val Gly Tyr Ser Ser Ser Ser Ala Leu Met Thr ThrLeu Thr Gln Asn 275 280 285 Ala Ser Ser Ser Ala Ala Asp Ser Arg Ser GlyArg Lys Ser Lys Asn 290 295 300 Asn Asn Lys Ser Ser Ser Gln Gln Ser SerSer Ser Ser Ser Ser Ser 305 310 315 320 Ser Leu Ser Ser Cys Ser Ser SerSer Thr Val Val Gln Glu Ile Ser 325 330 335 Gln Gln Thr Thr Val Val ProGlu Ser Asp Ser Asn Ser Gln Val Asp 340 345 350 Trp Thr Tyr Asp Pro AsnGlu Pro Arg Tyr Cys Ile Cys Asn Gln Val 355 360 365 Ser Tyr Gly Glu MetVal Gly Cys Asp Asn Gln Asp Cys Pro Ile Glu 370 375 380 Trp Phe His TyrGly Cys Val Gly Leu Thr Glu Ala Pro Lys Gly Lys 385 390 395 400 Trp TyrCys Pro Gln Cys Thr Ala Ala Met Lys Arg Arg Gly Ser Arg 405 410 415 HisLys 2 1807 DNA Homo sapiens p47ING3 2 agcgggtgct gctagcggag gcgccatattggaggggaca aaactccggc gacagcgagt 60 gacacaaata aacccctgga cccccttgttccctcagctc taagggccgc gatgttgtac 120 ctagaagact atctggaaat gattgagcagcttcctatgg atctgcggga ccgcttcacg 180 gaaatgcgcg agatggacct gcaggtgcagaatgcaatgg atcaactaga acaaagagtc 240 agtgaattct ttatgaatgc aaagaaaaataaacctgagt ggagggaaga gcaaatggca 300 tccatcaaaa aagactacta taaagctttggaagatgcag atgagaaggt tcagttggca 360 aaccagatat atgacttggt agatcgacacttgagaaagc tggatcagga actggctaag 420 tttaaaatgg agctggaagc tgataatgctggaattacag aaatattaga gaggcgatct 480 ttggaattag acactccttc acagccagtgaacaatcacc atgctcattc acatactcca 540 gtggaaaaaa ggaaatataa tccaacttctcaccatacga caacagatca tattcctgaa 600 aagaaattta aatctgaagc tcttctatccacccttacgt cagatgcctc taaggaaaat 660 acactaggtt gtcgaaataa taattccacagcctcttcta acaatgccta caatgtgaat 720 tcctcccaac ctctgggatc ctataacattggctcgttat cttcaggaac tggtgcaggg 780 gcaattacca tggcagctgc tcaagcagttcaggctacag ctcagatgaa ggagggacga 840 agaacatcaa gtttaaaagc cagttatgaagcatttaaga ataatgactt tcagttggga 900 aaagaatttt caatggccag ggaaacagttggctattcat catcttcggc acttatgaca 960 acattaacac agaatgccag ttcatcagcagccgactcac ggagtggtcg aaagagcaaa 1020 aacaacaaca agtcttcaag ccagcagtcatcatcttcct cctcctcttc ttccttatca 1080 tcgtgttctt catcatcaac tgttgtacaagaaatctctc aacaaacaac tgtagtgcca 1140 gaatctgatt caaatagtca ggttgattggacttacgacc caaatgaacc tcgatactgc 1200 atttgtaatc aggtatctta tggtgagatggtgggatgtg ataaccaaga ttgccctata 1260 gaatggttcc attatggctg cgttggattgacagaggcac caaaaggcaa atggtactgt 1320 ccacagtgca ctgctgcaat gaagagaagaggcagcagac acaaataaag gtggtccttt 1380 tgtttgatga agaaataaac ttcagctgaagattttatat aggactttaa aaagaagaga 1440 agagaaagaa gaaacaatgc atttccaggcaaccacttaa aggatttaca tagacaatcc 1500 tataagatct tgaacttgaa ttttatgggttgtattttaa taatgtaagt aaattattta 1560 tgcactcctg gtgtgctatg aatattattccagttagcct tggattattt cagtggccaa 1620 catatgcaga catttgtact cctcaaccattttctcaaag taatgggcat tctatgattt 1680 agacttcaag gaattccaat gatgaagattttaaggaaag tattttatat tcaacaggta 1740 tattctgctg catgtactgt actccagagctgttatgtaa cactgtatat aaatggttgc 1800 aaaaaaa 1807 3 7 PRT Homo sapiensamino acids 1-7 of p47ING3 encoded by degenerate primers 3 Met Leu TyrLeu Glu Asp Tyr 1 5 4 7 PRT Homo sapiens amino acids 412-418 encoded byp47ING3 degenerate primers 4 Arg Arg Gly Ser Arg His Lys 1 5 5 17 PRTHomo sapiens peptide 141-157 of p47ING3 (ping-3) 5 His Thr Pro Val GluLys Arg Lys Tyr Asn Pro Thr Ser His His Thr 1 5 10 15 Thr 6 280 PRT Homosapiens p33ING2 6 Met Leu Gly Gln Gln Gln Gln Gln Leu Tyr Ser Ser AlaAla Leu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu Thr Cys Tyr ValGln Asp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His Asp Met Gln ArgAsn Val Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr Gln Glu Thr LeuLys Glu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys Glu Asp Asp LeuAsn Gln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln Arg Ala Leu IleAsn Ser Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile Val Thr Gln MetLeu Glu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met Glu Leu His SerGln Cys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg Ala Ser Asp LysAla Lys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser Ser Arg Arg ProArg Arg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp Leu Cys His MetAla Asn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro Pro Lys Glu LysLys Ser Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 Lys Ala Lys GlnGlu Arg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205 Pro Asn GluPro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215 220 Met IleGly Cys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225 230 235 240Ser Cys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr Cys Pro 245 250255 Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser Thr Glu Lys 260265 270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 7 1080 DNA Homo sapiensp33ING2 7 gcggccgcgg ccggtgcatg tgcggctgct ggatgcggag gcggcggcgacggcgcggat 60 cggcaggatg ttagggcagc agcagcagca actgtactcg tcggctgcgctcctgaccgg 120 ggagcggagc cggctgctca cctgctacgt gcaggactac cttgagtgcgtggagtcgct 180 gccccacgac atgcagagga acgtgtctgt gctgcgagag ctggacaacaaatatcaaga 240 aacgttaaag gaaattgatg atgtctacga aaaatataag aaagaagatgatttaaacca 300 gaagaaacgt ctacagcagc ttctccagag agcactaatt aatagtcaagaattgggaga 360 tgaaaaaata cagattgtta cacaaatgct cgaattggtg gaaaatcgggcaagacaaat 420 ggagttacac tcacagtgtt tccaagatcc tgctgaaagt gaacgagcctcagataaagc 480 aaagatggat tccagccaac cagaaagatc ttcaagaaga ccccgcaggcagcggaccag 540 tgaaagccgt gatttatgtc acatggcaaa tgggattgaa gactgtgatgatcagccacc 600 taaagaaaag aaatccaagt cagcaaagaa aaagaaacgc tccaaggccaagcaggaaag 660 ggaagcttca cctgttgagt ttgcaataga tcctaatgaa cctacatactgcttatgcaa 720 ccaagtgtct tatggggaga tgataggatg tgacaatgaa cagtgtccaattgaatggtt 780 tcacttttca tgtgtttcac ttacctataa accaaagggg aaatggtattgcccaaagtg 840 caggggagat aatgagaaaa caatggacaa aagtactgaa aagacaaaaaaggatagaag 900 atcgaggtag taaaggccat ccacatttta aagggttatt tgtcttttatataattcgtt 960 tgctttcaga aaatgtttta gggtaaatgc ataagactat gcaataatttttaatcatta 1020 gtattaatgg tgtattaaaa gttgttgtac tttgaaaaaa aaaaaaaaaaaaaaaaaaaa 1080 8 279 PRT Homo sapiens p33ING1 8 Met Leu Ser Pro Ala AsnGly Glu Gln Leu His Leu Val Asn Tyr Val 1 5 10 15 Glu Asp Tyr Leu AspSer Ile Glu Ser Leu Pro Phe Asp Leu Gln Arg 20 25 30 Asn Val Ser Leu MetArg Glu Ile Asp Ala Lys Tyr Gln Glu Ile Leu 35 40 45 Lys Glu Leu Asp GluCys Tyr Glu Arg Phe Ser Arg Glu Thr Asp Gly 50 55 60 Ala Gln Lys Arg ArgMet Leu His Cys Val Gln Arg Ala Leu Ile Arg 65 70 75 80 Ser Gln Glu LeuGly Asp Glu Lys Ile Gln Ile Val Ser Gln Met Val 85 90 95 Glu Leu Val GluAsn Arg Thr Arg Gln Val Asp Ser His Val Glu Leu 100 105 110 Phe Glu AlaGln Gln Glu Leu Gly Asp Thr Ala Gly Asn Ser Gly Lys 115 120 125 Ala GlyAla Asp Arg Pro Lys Gly Glu Ala Ala Ala Gln Ala Asp Lys 130 135 140 ProAsn Ser Lys Arg Ser Arg Arg Gln Arg Asn Asn Glu Asn Arg Glu 145 150 155160 Asn Ala Ser Ser Asn His Asp His Asp Asp Gly Ala Ser Gly Thr Pro 165170 175 Lys Glu Lys Lys Ala Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala180 185 190 Lys Ala Glu Arg Glu Ala Ser Pro Ala Asp Leu Pro Ile Asp ProAsn 195 200 205 Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly GluMet Ile 210 215 220 Gly Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Phe HisPhe Ser Cys 225 230 235 240 Val Gly Leu Asn His Lys Pro Lys Gly Lys TrpTyr Cys Pro Lys Cys 245 250 255 Arg Gly Glu Asn Glu Lys Thr Met Asp LysAla Leu Glu Lys Ser Lys 260 265 270 Lys Glu Arg Ala Tyr Asn Arg 275 9 10PRT Homo sapiens peptide 1-10 of p47ING3 (Ping-3N peptide) 9 Met Leu TyrLeu Glu Asp Tyr Leu Glu Met 1 5 10

What is claimed is:
 1. A method for identifying a p47ING3 modulatingcompound, the method comprising: contacting a cell expressing p47ING3above basal levels with a test compound; and determining whether saidtest compound is able to modulate a p47ING3 activity.
 2. The method ofclaim 1, wherein said p47ING3 activity is selected from the groupconsisting of: a p47ING3 mediated cell-cycle arrest, a p47ING3 inducedchange in cell growth, and a p47ING3 mediated decrease of colonyformation.
 3. The method of claim 1, wherein said p47ING3 polypeptide isrecombinant.
 4. The method of claim 1, wherein said p47ING3 polypeptideis from a human.
 5. The method of claim 1, wherein said p47ING3polypeptide has an amino acid sequence of SEQ ID NO:1.
 6. A method ofinhibiting cellular proliferation, the method comprising: increasing theamount of p47ING3 polypeptide in a cell, wherein said cell produces awild-type p53 molecule.
 7. The method of claim 6, wherein said increasein the amount of p47ING3 is achieved by a method selected from the groupconsisting of: tranducing said cell with a p47ING3 expression cassette,activating the promoter of the endogenous p47ING3 gene in said cell witha DNA activation sequence, and microinjecting a p47ING3 protein moleculeinto said cell.
 8. The method of claim 6, wherein said p47ING3polypeptide comprises a sequence of SEQ ID NO:1.
 9. The method of claim6, wherein said p47ING3 polypeptide is encoded by a nucleotide sequenceof SEQ ID NO:2.
 10. The method of claim 6, wherein said p47ING3polypeptide is from a human.
 11. The method of claim 6, wherein saidp47ING3 polypeptide has a molecular weight of about 40 kDa to about 47kDa.
 12. The method of claim 6, wherein said cell is an RKO human coloncancer cell.
 13. A method of detecting the presence or absence ofp47ING3 in tumorigenic mammalian tissue, the method comprising the stepsof: (i) isolating a tumorigenic sample; (ii) contacting the tumorigenicsample with a p47ING3-specific reagent that selectively associates withp47ING3; and (iii) detecting the level of p47ING3-specific reagent thatselectively associates with the tumorigenic sample.
 14. The method ofclaim 13, wherein the p47ING3-specific reagent is selected from thegroup consisting of a p47ING3-specific antibody, a p47ING3-specificprimer, and a p47ING3-specific nucleic acid probe.
 15. The method ofclaim 14, wherein the p47ING3-specific nucleic acid probe binds to anucleic acid sequence to a nucleic acid of SEQ ID NO:2.
 16. The methodof claim 13, wherein the tumorigenic sample comprises intact chromosome7q31.
 17. The method of claim 13, wherein the p47ING3-specific reagentdetects nucleic acid.
 18. The method of claim 17, wherein the nucleicacid is RNA.
 19. The method of claim 13, wherein the p47ING3-specificreagent is an antibody that selectively binds to p47ING3.
 20. The methodof claim 13, wherein the antibody is polyclonal.
 21. An isolated nucleicacid encoding a tumor suppressor polypeptide (p47ING3), the nucleic acidcomprising SEQ ID NO:
 2. 22. An expression vector comprising SEQ ID NO:2.
 23. A host cell transfected with the vector of claim
 22. 24. Anisolated tumor suppressor polypeptide (p47ING3), the polypeptide: havingan amino sequence of SEQ ID NO:1.
 25. An antibody that selectively bindsto a p47ING3 polypeptide of SEQ ID NO:
 1. 26. The antibody of claim 12,wherein the antibody is polyclonal.